Bile acids promote diethylnitrosamine-induced hepatocellular carcinoma via increased inflammatory signaling

Abstract

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1β mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Keywords: NF-κB; Nanog; cholic acid; proliferation.

Fig. 1.

Cholic acid (CA) promotes diethylnitrosamine (DEN)-induced hepatic carcinogenesis. A: scheme showing the experimental design. B: representative photographs of mouse liver obtained from DEN + normal diet (ND) and DEN + 0.2% CA treated mice. Arrows point to liver tumors. C: bar graph showing number of liver tumors per mouse. D: percent survival in various treatment groups. Data are expressed as mean ± SE. *P < 0.05, significant difference from all other groups. E: representative photomicrographs of hemtoxylin-eosin (H&E)-stained liver sections from ND (i), 0.2% CA (ii), DEN + ND (iii), and DEN + 0.2% CA (iv) taken at ×400 magnification. Arrowheads demark the tumor boundary and arrows point to inflammatory cell infiltrate.

Fig. 2.

Histological characterization of hepatocellular carcinoma (HCC). A: representative photomicrographs of H&E-stained sections showing globular inclusions (i, arrowheads) and immunohistochemical detection of ubiquitin (ii) and CK8 (iii). B: representative photomicrographs of CD34 immunohistochemistry on liver sections from ND (i) and DEN + 0.2% CA (ii) treated mice. Liver section from DEN + 0.2% CA treated mouse showing vascular invasion of an HCC. iii: H&E staining (×200). iv: CD34 immunohistochemistry (×200). *Vascular invasion. C: representative photomicrographs of reticulin staining in ND (i) and DEN + 0.2% CA treated mice (ii: ×400. iii: ×200). *HCC within the section and arrowheads show tumor boundary. D: representative photomicrographs of CD3 (i), CD45 (ii), and CD20 (iii). All photographs at ×400 magnification unless mentioned otherwise.

Fig. 3.

Analysis of cell proliferation and apoptosis in CA-promoted DEN-induced HCC. A: representative photomicrographs of PCNA immunohistochemistry performed on paraffin embedded liver sections from various treatment groups. Arrows indicate cells in S phase of cell cycle. B: quantification of PCNA staining. Bar graph shows numbers of PCNA-positive cells in high power (×400) fields in various treatments groups. Data are expressed as mean ± SE. *P < 0.05, significant difference from ND. C: Western blot analysis of cyclin D1, PCNA, and p21 conducted using total liver cell extracts. D: representative photomicrographs of terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay immunofluorescent staining on liver sections from ND (i), 0.2% CA (ii), DEN + ND (iii), and DEN + 0.2% CA (iv). White arrowheads point to cells in apoptosis and yellow arrows point to necrotic cells. E: bar graph showing quantification of TUNEL assay performed using paraffin embedded liver sections from various treatment groups. Data are expressed as mean ± SE. *P < 0.05, significant difference from ND. !P < 0.5, significantly different from DEN + ND group. F: Western blot analysis of total and activated caspase-3 conducted using total liver extracts from various treatment groups.

Fig. 4.

Increased inflammation in CA-promoted DEN-induced HCC. Real-time PCR analysis of TNF-α (A) and IL-1β mRNA (B) in various groups. Data are expressed as mean ± SE. *P < 0.05, significant difference from the ND group. !P < 0.05, significant difference from all other groups. Western blot analysis of total p65, total and phosphorylated IκBα (C) and total and phosphorylated IκKα/β (D) conducted using total liver extracts from various groups.

Fig. 5.

Changes in MAPK signaling in CA-promoted DEN-induced HCC. Western blot analysis of total and phosphorylated JNK and AKT (A) and total and phosphorylated ERK and p38 MAPK (B) conducted using total liver extracts from various groups. C–F: densitometric analysis of Western blots of JNK, AKT, ERK, and p38, respectively, conducted using blots shown in A and B.

Fig. 6.

Changes in bile acids in serum and liver during CA-induced promotion of DEN-initiated tumors. A: quantification of total bile acids in serum in mice after various treatment groups. B–E: bar graphs showing contribution of each bile acid to total bile acids in serum of ND, 0.2% CA, DEN + ND, and DEN + 0.2% CA groups. F: quantification of total bile acids in livers of mice after various treatment groups. G–J: bar graphs showing contribution of each bile acid to total bile acids in serum of ND, 0.2% CA, DEN + ND, and DEN + 0.2% CA groups. Data are expressed as mean ± SE. In B–E, all comparisons are made to respective bile acid concentration in ND group. *P < 0.05, significant difference. **P < 0.01, significant difference.

Fig. 7.

Bile acids induce stemness genes in mouse hepatocytes. Bar graphs showing mRNA levels of Myc, KLF4, Nanog, Oct4, and Sox2 as measured by Rea Time PCR using total RNA extracted from primary mouse hepatocytes treated with various bile acids for 24 h. Data are expressed as mean ± SE. *P < 0.05, significant difference. **P < 0.01, significant difference. ***P < 0.001, significant difference.

Fig. 8.

Serum total bile acids in normal, cirrhotic and HCC patient samples. Data are expressed as mean ± SE. ***P < 0.001, significant difference.